Lung cancer is the most common cause of cancer deaths worldwide, with one of every three cancer-related deaths attributable to lung cancer. As most current and former smokers have small (< 2 mm) nodules, a sound chemoprevention strategy would be to prevent their further growth by targeting angiogenesis. Consequently, mechanism-based angioprevention approaches employing animal models are realistic strategies to develop tools for combating lung cancer. Completed studies by us show that feeding silibinin, the major active constituent in milk thistle extract, a widely consumed dietary supplement, to A/J mice results in a 38-46% inhibition in lung tumor number and up to 93% inhibition of urethane-induced lung tumor growth. Immunohistochemical (IHC) analyses of the lung tumors showed strong inhibition of CD31 and iNOS staining in silibinin fed compared to urethane alone mice. Based on these studies and the fact that iNOS plays an important role in both angiogenic and extra-angiogenic (e.g. proliferation and apoptosis) mechanisms, we hypothesize that silibinin is a novel non-toxic agent that targets iNOS regulation in its angiopreventive efficacy against lung tumorigenesis. To test this hypothesis, our specific aims are: 1) To further assess and establish iNOS regulation and angioprevention in silibinin efficacy against urethane-induced lung tumorigenesis, 2) To assess and establish whether iNOS is the sole target of silibinin's mechanism of ameliorating mouse lung tumorigenesis, or whether an alternate/additional pathway also exists, 3) To identify and define the mechanisms by which silibinin modulates iNOS levels in lung cancer cells, and to establish the in vivo significance of these mechanisms in the overall anti-tumor efficacy of silibinin, and 4) To identify and define the role of iNOS regulation by silibinin in its overall angiopreventive efficacy, employing endothelial and lung cancer cell cultures. We anticipate that these proposed studies, together with our earlier work, will identify silibinin as a mechanism-based agent for the prevention and growth control of lung cancer. This will establish its in vivo efficacy in pre-clinical carcinogenic and genetic models. As a practical and translational approach, the long range goal of these studies is to define and establish the usefulness of silibinin for preventing and controlling human lung cancer growth. Accordingly, completion of the studies proposed in this grant would position us for a pilot clinical trial with silibinin in lung cancer patients and in those suspected of being at high risk for lung cancer development.